The normal hemostatic system limits bleeding and thrombosis by precisely regulated interactions between components of the blood vessel wall, circulating blood platelets and plasma proteins (see, e.g., Harrison's "Principles of Internal Medicine" (1997) 14.sub.th Edition, Fauci et al. (eds.), Mc Graw Hill, New York). Disruption in either of the two processes of the hemostatic system--clot formation and clot lysis--can have severe medical repercussions. Hemorrhage, thrombosis and embolism are all common clinical manifestations of many diseases. Thus, the ability to efficiently screen a large number of candidate agents for their ability to modulate clot formation, or clot lysis (thrombolysis) would greatly increase the therapeutic repertory currently available to treat such conditions.
The clotting cascade of the hemostatic process is initiated when trauma, surgery or disease disrupt the vascular endothelial lining and blood is exposed to subendothelial connective tissue. The injured vascular endothelial cell releases substances that initiate the clotting cascade, a process involving the activation of a series of compounds that ultimately results in the conversion of prothrombin to thrombin. Thrombin is a key enzyme in the coagulation event, catalyzing the activation of platelets and the cleavage of fibrinogen.
While formation of fibrin monomers provides the structural matrix of a clot, aggregation of platelets is critical for the clot's integrity. Upon activation by thrombin, the platelet expresses on its surface the IIb/IIIa receptor, which allows for the binding of von Willebrand Factor. The binding of von Willebrand Factor to the IIb/IIIa receptor of adjacent platelets results in the platelet aggregation reaction.
Platelets are small disc-shaped cell fragments formed by the breakdown of megakaryocytes, and are very susceptible to changes in their environment such as pH and temperature. In normal blood banking procedures, donated whole blood is stored at a temperature of approximately 4.degree. C. Upon refrigeration, the platelet irreversibly changes its structural conformation from a disc shape to a sphere. When the platelet is not in the normal disc shape, it is incapable of aggregation and thus not physiologically relevant. In addition to the activation of platelets, thrombin also acts on fibrinogen, the key structural protein in blood clot formation and the substrate for thrombin proteolytic activity. Thrombin catalyzes the release of small peptides, fibrinopeptides A and B, from the chains of fibrinogen. The removal of the fibrinopeptides from the fibrinogen substrate results in the formation of fibrin monomers which polymerize into fibers and provide the structural matrix of the clot. The clot formed by fibrin is removed or degraded by the process of fibrinolysis. Fibrinolysis is initiated by the release of either tissue plasminogen activator or prourokinase from endothelial cells. These agents convert plasminogen into the active proteolytic enzyme, plasmin which catalyzes the fibrin substrate into soluble degradation products. In addition, plasmin is enzymatically active against fibrinogen, and degrades fibrinogen into soluble products.
Various methods for assaying candidate agents that modulate the formation, inhibition, or degradation of clots have been described previously (Taylor et al. (1973) Ser. Haemat. VI:528; Krishnamurti et al. (1994) Thrombosis Research 73:419; Charlton et al. (1996) Thrombosis and Haemostiasis 75:808). However, previously known assays only utilized blood isolated from on-site donors, either human or animal. Consequently, the scale and frequency of candidate agents to be screened were limited by the availability of on-site donors. Thus, there is a significant need for a physiologically relevant, rapid, and inexpensive assay to determine the potential of candidate drugs to modulate clot formation or clot lysis. The subject application provides such a method.